The goal of this research is to contribute to an understanding of the structural and functional organization of the eukaryotic chromosome, especially in relation to the process of DNA replication. Most experiments will involve yeast, a simple eukaryote. The nature of initiation sites for DNA replication will be studied in a number of ways. DNAs from a variety of eukaryotes will be tested for their ability to replicate autonomously in yeast. Those DNAs capable of autonomous replication will be subcloned in order to characterize the sequences responsible for replicator activity. DNAs being studied in this way include mitochondrial DNA from Xenopus and rat, chicken alpha and beta globin DNAs, and human middle repetitive sequences. Initiation of DNA replication will also be studied using the naturally occurring yeast plasmid 2-micron DNA. We will investigate how reactivation of the origin of replication of this plasmid is prevented in a given S-phase. We are also attemping to select for yeast cells capable of expressing the thymidine kinase gene of Herpes Simplex I in order to obtain a strain of yeast which can be isotopically labeled with thymidine.